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Gas Chromatography

Lecture 40

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Liquid Stationary Phases

In general, the polarity of the stationary phase should match that of the sample constituents ("like" dissolves "like"). Most stationary phases are based on polydimethylsiloxane or polyethylene glycol (PEG) backbones:

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The polarity of the stationary phase can be changed by derivatization with different functional groups such as a phenyl group. Bleeding of the column is cured by bonding the stationary phase to the column; or crosslinking the stationary phase.

Liquid Stationary Phases should have the following characteristics:

• Low volatility• High decomposition

temperature (thermally stable)

• Chemically inert (reversible interactions with solvent)

• Chemically attached to support (to prevent bleeding)

• Appropriate k' and for good resolution

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Bonded and Crosslinked Stationary Phases

The purpose of bonding and cross-linking is to prevent bleeding and provide a stable stationary phase. With use at high temperatures, stationary phases that are not chemically bonded or crosslinked slowly lose their stationary phase due to bleeding in which a small amount of the physically immobilized liquid is carried out of the column during the elution process. Crosslinking is carried out in situ after a column is coated with one of the polymers

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In summary, stationary phases are usually bonded and/or crosslinked and the following remarks are usually helpful:

1. Bonding occurs through covalent linking of stationary phase to support

2. Crosslinking occurs through polymerization reactions to join individual stationary phase molecules

3. Nonpolar stationary phases are best for nonpolar analytes where nonpolar analytes are retained preferentially

4. Polar stationary phases are best for polar analytes where polar analytes are retained preferentially

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The thickness of the stationary phase affects the performance of the column as follows:

1. Increasing thickness of stationary phase allows the separation of larger sample sizes.

2. Increasing thickness of stationary phase reduces efficiency since HS increases.

3. Increasing thickness of stationary phase is better for separation of highly volatile compounds due to increased retention.

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Temperature Programming

Gas chromatographs are usually capable of performing what is known as temperature programming gas chromatography (TPGC). The temperature of the column is changed according to a preset temperature isotherm. TPGC is a very important procedure, which is used for the attainment of excellent looking chromatograms in the least time possible. For example, assume a chromatogram obtained using isothermal GC at 80 oC, as shown below:

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The General Elution ProblemLook at the chromatogram below in which six

components are to be separated by an elution process using isothermal conditions at for example 120 oC:

 

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It is clear from the figure that the separation is optimized for the elution of the first two components. However, the last two components have very long retention and appear as broad peaks. Using isothermal conditions at high temperature (say for example 200oC) can optimize the elution of the last two compounds but, unfortunately, results in bad resolution of the earlier eluting compounds as shown in the figure below where the first two components are coeluted while the resolution of the second two components becomes too bad:

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One can also optimize the separation of the middle too components by adjusting the isothermal conditions (for example at say 160 oC). In this case, a chromatogram like the one below can be obtained:

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However, in chromatographic separations we are interested in fully separating all components in an acceptable resolution. Therefore, it is not acceptable to optimize the separation for a single component while disregarding the others. The solution of this problem can be achieved by consecutive optimization of individual components as the separation proceeds. In this case, temperature should be changed during the separation process. This is called temperature programming gas chromatography (TPGC)

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First, a temperature suitable for the separation of the first eluting component is selected, and then the temperature is increased so that the second component is separated and so on. The change in temperature can be linear, parabolic, step, or any other formula. The chromatographic separation where the temperature is changed during the elution process is called TPGC. A separation like the one below can be obtained:

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Interfacing GC with other Methods

As mentioned previously, chromatographic methods (including GC) use retention times as markers for qualitative analysis. However, this characteristic does not absolutely confirm the existence of a specific analyte as many analytes may have very similar stationary phases. GC, as other chromatographic techniques, can confirm the absence of a solute rather than its existence. When GC is coupled with structural detection methods, it serves as a powerful tool for identifying the components of complex mixtures. A popular combination is GC/MS.

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194

67 109

5582

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16513694

40 60 80 100 120 140 160 180 200

Abundance

Mass (amu)

Mass Spectrum

NC

CNH

CO

C

O

N

N

C H

C 3H

C3H

MassSpectrometer

Mass Spectrometry

Typical sample: isolatedcompound (~1 nanogram)

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HPLC

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Four main chromatographic techniques that use a liquid mobile phase are covered under the broad high performance liquid chromatographic technique. These include:

1. Partition Chromatography2. Liquid-Solid Chromatography3. Ion-Exchange Chromatography4. Size Exclusion (Gel Permeation)

ChromatographyThe first of the abovementioned chromatographic

technique is most widely used. Generally, HPLC uses very high pressures (up to 4000 psi) and very small particle size (down to 3 mm).

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Column efficiency in Liquid Chromatography

We have seen earlier that several factors affect efficiency in chromatographic techniques including:

1. Particle size

2. Flow rate

3. Thickness of stationary phase

4. Mobile phase viscosity

5. Diffusion of solute in mobile and stationary phases

6. How well a column is packed

7. Sample size (mg sample/g packing)

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Extra-Column Band Broadening

We have discussed three reasons for intra column band broadening including:

1. Multiple paths effects

2. Longitudinal diffusion

3. mass transfer in stationary and mobile phases

However, there are other sources of band broadening unrelated to column materials and occur outside the column. These include :

1. Fittings dead volume

2. Tubing length and diameter

3. Detector volume

4. Injection volume

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Instruments for Liquid ChromatographyPumpsThree types of pumps are known:1. Reciprocating pumps2. Displacement Pumps3. Pneumatic pumps4. Reciprocating pumps are by far the most

widely used and practically 100% of the pumps used in commercial HPLC equipment are of the reciprocating type.

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Reciprocating pumps

In reciprocating pumps, a motor driven reciprocating piston controls the flow of mobile phase with the help of two ball check valves that opens and closes with the piston movement. The flow is thus not continuous and damping of flow is necessary. This is accomplished using pulse dampers which are a long coiled capillary tube.

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